Engraftment of wild-type alveolar type II epithelial cells in surfactant protein C deficient mice

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cell therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model, Sftpc−/− mice, to provide proof-of-principle for this approach. Sftpc−/− mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to two months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD-like disease.

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Supportive therapy, such as corticosteroids, hydroxychloroquine, and azithromycin has limited benefit for the majority of cases leaving lung transplantation as the only definitive treatment 6,9,28 .Lung transplantation remains limited by the shortage of suitable donor organs, especially for smaller children 29,30 .Moreover, the overall survival rate at five years post-transplant is just 55%, a figure that has not significantly improved in the last 20 years 30 .There is therefore an urgent need for improved treatments.
Cell therapy, in which gene-corrected cells are transplanted into the lungs to replace defective ATII cells, is an exciting treatment option for chILD.In rats, intratracheal (i.t.) administration of ATII cells after bleomycin injury may result in reduction of fibrosis 31,32 .
In mice infected with H1N1 influenza, oxygen saturation improved after ATII cell transplant 33 .It is unknown whether transplanted ATII cells could affect the disease, and if so, how many endogenous cells need to be replaced with exogenous ATII cells for therapeutic effect.
We investigated the efficacy of cell therapy in a viable mouse model of chILD-like disease, the Sftpc -/-mouse.In humans, SFTPC encodes a 191 or 197 amino acid protein (length varies due to alternative splicing) that is palmitoylated, proteolytically processed in the endoplasmic reticulum, and shuttled to the multivesicular bodies where final cleavage generating mature SPC (35 amino acids) takes place.SPC is then incorporated into LBs in which surfactant is stored prior to secretion in the alveolar space 2,34 .Clinically, mutations in SFTPC have variable penetrance with phenotypes ranging from neonatal respiratory failure to chILD to idiopathic pulmonary fibrosis in adulthood.Some patients require lung transplant, others are managed with long-term mechanical ventilation, and others remain almost asymptomatic.Postnatal mortality is rare 35,36 .The most common human SFTPC mutation to cause chILD is the threonine-to-isoleucine substitution at the codon 73 (SFTPC I73T ) resulting in mis-trafficking of SPC with proteotoxicity 37 .One study has demonstrated successful in utero gene editing using a mouse model of the most common SFPTC mutation responsible for chILD, Sftpc I73T . 38To be clinically applicable, diagnosis and correction have to occur before birth, which is not yet feasible 38 .
While our animal model is a double knockout which is not a genetic mimic of the most common pathogenic human SFTPC mutation, it recapitulates three critical characteristics of chILD: lack of normal surfactant, fibrosis, progressive lung disease with age.
In this proof-of-concept study, we used Sftpc -/-mice to show, for the first time, an effective cell therapy in an animal model of chILD-like disease.

Results
Morphological changes of the lung in Sftpc -/-mice.Previous work showed that Sftpc -/- mice on a 129/Sv background developed an interstitial pneumonitis-like phenotype beginning at two months of age and worsening with age up to 12-14 months 39 .The phenotype was characterized by irregular alveolar septation, mononucleated cell infiltrates, ATII cell hyperplasia, and interstitial thickening 39 .To develop a time course of the disease, we analyzed the lung histology and stereology in Sftpc -/-(knock out, KO) and Sftpc +/+ (wild-type, WT) mice at 4-, 8-, and 12-month old (mo) (Figure 1).Histological analysis of lung sections showed progressive lung injury characterized by neutrophil infiltration into interstitium and airspace, alveolar wall thickening, and appearance of proteinaceous debris in the alveolar space.These features worsened with age in Sftpc -/-but not in Sftpc +/+ mice (Figure 1a).For the pathological evaluation of lung injury score (LIS), consistent with the American Thoracic Society (ATS) criteria for lung injury 40,41 , lung fields were randomly selected using a standardized digital method recently established 42 .LIS was significantly higher (worse) in Sftpc -/-mice compared to age-matched Sftpc +/+ mice, where no changes over age were noted (Figure 1b; Supplementary Figure 1a and b).Alveolar septa count (number of interalveolar septa), volume density of alveolar septa (an estimation of alveolar airspace), mean trans-sectional wall length (a measure of alveolar septal thickness), airspace surface area density (estimation of lung volume), but not mean linear intercept (estimation of volume to surface ratio of the airspace), were all significantly altered in Sftpc -/-mice with age (Figure 1c) and overall different from those of the corresponding Sftpc +/+ mice (Figures 1c and Supplementary Figure 1c), supporting the presence of a fibrotic remodeling process in Sftpc -/-mice consistent with prior data 39,43 .The presence of interstitial thickening was further supported by elevated deposition of collagen (col) I and col IV (Figure 2a and Supplementary Figure 2a), and a significant agedependent increased hydroxyproline [(2S,4R)-4-hydroxyproline, Hyp] content in Sftpc - /-compared to Sftpc +/+ lungs (Figure 2b and Supplementary Figure 2b).KO mice, lacking Sftpc and the related protein SPC, (Figure 2c-d), did not show a difference in the mRNA expression of either other surfactant genes (Sftpa, Stfpb, Sftpd) (Figure 2d) or other ATII cell markers, Lysosomal Associated Membrane Protein 3 (Lamp3) and Abca3 (Figure 2c and 2e).Taken together, our data indicate that lack of SPC results in morphological and stereological changes similar to histological patterns observed in chILD and worsening with age, while other ATII markers and surfactant genes do not appear clearly affected.
Increased susceptibility to bleomycin in Sftpc -/-mice.We next explored whether bleomycin could be used to partially ablate endogenous defective ATII cells in Sftpc -/- mice.Sftpc -/-mice on a Black Swiss background have normal lung structure at baseline 44 , but develop fibrosis after a low-dose (0.01U/mouse) of bleomycin compared to Sftpc +/+ mice 45 .In contrast, Sftpc -/-mice on 129Sv background, described here, show interstitial pneumonitis at baseline (Figure 1 and 39 ) and develop extensive disruption of lung architecture and persistent lung inflammation after 0.05 U of bleomycin/mouse (a dose commonly used in Sftpc +/+ mice) 46 .We administered incremental doses of bleomycin (0.005, 0.01, and 0.05 U/mouse) i.t. to 4 mo Sftpc -/-and Sftpc +/+ mice (Figure 3a).After 10 days, Sftpc -/-mice showed increased alveolar wall thickening, interstitial neutrophils, hyaline membrane and proteinaceous debris lining alveolar walls at any dose of bleomycin while Sftpc +/+ mice showed initial histological evidence of lung injury at 0.01 U of bleomycin and clearly visible only at 0.05 (Figure 3b and Supplementary Figure 3).
Compared to Sftpc +/+ mice, Sftpc -/-mice had significantly higher LIS at any dose (Figure 3c and Supplementary Figure 3).Stereological analysis confirmed a bleomycin dosedependent injury in Sftpc -/-mice for septal count, septal density, and mean transactional wall length but not for mean linear intercept and airspace surface density (Supplementary Figure 4).Nevertheless, we did notice that all the stereological values, except for septal counts, were significantly more altered in Sftpc -/-compared to Sftpc +/+ for each bleomycin dose (Supplementary Figure 4).Elevated deposition of collagens (I and IV) and quantification of HPO were observed only at the highest dose of bleomycin in the Sftpc -/-mice (Figure 4a-b and Supplementary Figure 5a).RT-qPCR analysis showed downregulation of mRNAs encoding Nkx2.1 as well as other ATII cell markers such as Abca3 with increasing bleomycin dose In the Sftpc -/-mice (Figure 4c and Supplementary Figure 5b).We conclude that Sftpc -/-mice are susceptible to bleomycin injury including its effect on ATII more than Sftpc +/+ .Engraftment of syngeneic Sftpc +/+ ATII cells in bleomycin-conditioned Sftpc -/-mice.We next investigated engraftment of ATII cells in bleomycin-treated Sftpc -/-mice.Primary ATII cells were isolated from Sftpc +/+ lungs, purified, and verified for epithelial markers and viability by fluorescence-activated cell sorting (FACS) (95-98% EPCAM + , CD45 -) and immunostaining for pro-SPC (Supplementary Figure 6a).4, 8, and 12 mo Sftpc -/-mice were conditioned with low-dose (0.005U/mouse) or high-dose (0.05U/mouse) bleomycin and 10 days later received 1x10 6 Sftpc +/+ ATII cells i.t.(Figure 5a).In preliminary experiments, 1x10 6 cells guaranteed consistent engraftment, 5x10 5 cells resulted in fewer engrafted cells, while 2x10 6 cells caused clumps of cells in the airways or inconsistent engraftment (data not shown).14 days post-transplantation, lungs were harvested and analyzed for cell engraftment (Figure 5a).By counting pro-SPC+ cells from immunofluorescent (IF) staining of representative lung sections, we noticed higher engraftment of Sftpc +/+ ATII cells in with low-dose bleomycin when compared to vehicle or to the high-dose (Figure 5b-d).Pro-SPC counterstained with RAGE (ATI cell marker) showed ATII cells in proximity of ATI cells in their physiologic and anatomic context (Figure 5c).
Supporting the pro-SPC immunostaining, we found increased expression of Sftpc mRNA in mice that received low-compared to high-dose bleomycin (Figure 5e).To further estimate the percentage of transplanted cells, genomic DNA (gDNA) was extracted from lung sections derived from transplanted mice and subject to RT-qPCR analysis for Sftpc using a methodology previously applied technique for other transplantation models 47 .
Using a standard curve of varying amounts of Sftpc +/+ cells alone (Supplementary Figure 6b-d) we estimated the engraftment of Sftpc +/+ cells per mouse.Again, we detected the higher number of transplanted Sftpc +/+ cells with low-dose bleomycin (Figure 5f).When we compare the engraftment efficient at different ages, we found that younger mice (4 and 8 mo) were engrafted more efficiently than older mice (12 mo) (Supplementary Figure 7).
Long-term engraftment and functionality of Sftpc +/+ ATII cells.Next, we asked if primary Sftpc +/+ ATII cells could promote repair.4 mo Sftpc +/+ mice were conditioned with lowdose bleomycin and 1x10 6 Sftpc +/+ ATII cells were delivered i.t. 10 days later, lungs were harvested and analyzed at 4 and 8 weeks post-transplantation.By immunostaining, we could detect a significant amount of pro-SPC+ ATII cells in the alveolar region of all the Sftpc -/-mice conditioned with bleomycin compared to those conditioned with vehicle only (Figure 6a).In transplanted mice, we could detect patches of LAMP3+ SPC+ ATII cells, indicating that transplanted Sftpc +/+ ATII cells were not only viable, but also capable to fully processing pro-SPC in its mature product, SPC (Figure 6b), as an indirect sign of their physiological recovery into the recipient lungs.By IF and mRNA and gDNA analysis for Sftpc, we confirmed the persistence of Sftpc +/+ cells up to 8 weeks (Figure 6c-e).
Finally, we asked whether transplanted ATII cells could promote repair of lungs injured by bleomycin.Histological analysis of mice lungs at 4 weeks post-transplantation suggested improved LIS when compared not only to mice conditioned with bleomycin but also to controls (vehicle and mock cell delivery) (Figure 6f).At 8 weeks, transplanted mice had an improved LIS compared to mice conditioned with bleomycin (Figure 6g).

Taken together, these data indicate that transplanted ATII cells can attenuate lung injury
induced by bleomycin and early they can also halt the disease progression.

Discussion
No prior examples of cell therapy have used an ATII cell disease model as in this case.
Using a chILD-like disease model, the 129/Sv Sftpc -/-model, we found that Sftpc +/+ ATII cells can engraft after pre-conditioning with low-dose bleomycin, especially early in the course of the disease, and engrafted Sftpc +/+ ATII cells are capable of repairing lung injury in those mice.
The initial study of Glasser and colleagues reported the generation of Sftpc knock-out mice on a Swiss black background 44 .Those mice were viable at birth and developed normally with only mild alterations in lung mechanics and decreased stability of surfactant when analyzed in vitro.In contrast, the same mutation on a 129/Sv background, used for the current studies, induced ATII hyperplasia with LB-like and lipid inclusions, increased neutrophils, alveolar macrophages with intracellular surfactant-like material, accumulation of a-smooth muscle actin (a-SMA) and are, therefore, a better model of the human disease 39 .Importantly, these mice are normal at birth, and progressively exhibit signs of interstitial fibrosis and cellular inflammation that progress with age with extensive remodeling, airspace loss and patchy fibrosis, and show low fertility rate 39 .Other animal models carrying Sftpc mutations exist, including a mouse expressing most common SFTPC mutation found in patients, SFTPC I73T . 17The Sftpc I73T mouse model , results in embryonic lethality unless inducibly expressed in the post-natal period 17,48 .After tamoxifen induction, these mice develop severe polycellular alveolitis with increased mortality between 7 and 14 days.This is in direct contrast with children affected by chILD-SFTPC I73T , in whom symptoms appear variably after birth and not typically associated with postnatal mortality 49,50 .Other variants of SFTPC in humans include L188Q, Dexon4, and C121G, all localized to the pro-SPC COOH-terminal (BRICHOS) domain 18,51 .They cause protein misfolding, aggregation, ER stress and apoptosis 18,51 .Mice expressing Dexon4 or the C121G SFTPC mutant show ATII cell death, fibrotic remodeling, and neonatal lethality 52,53 .When C121G mutant is expressed in an inducible manner, mice show a pattern of chronic fibrosis 40 .The mouse expressing L188Q and a recent knock-in model expressing this mutation, do not develop lung fibrosis unless conditioned with bleomycin 54,55 .In contrast to these inducible models, the model used in the current study showed a consistent phenotype across the experiments (Figure 1 and 2).Our model is therefore currently the best approximation of a pre-clinical model for cell therapy of chILD.
Bleomycin remains the most frequently used agent to induce pulmonary fibrosis in animal models.The fibrotic response of Sftpc -/-mice to bleomycin has already investigated in two previous studies 45,46 .Here instead, low-dose bleomycin was used as a conditioning strategy to partially depleting ATII cells prior to cell transplant.In fact, 0.005U/kg of bleomycin (one-tenth of the dose normally used in wild-type mice) was sufficient to partially affect ATII cells without severely further damaging lungs (Figures 3    and 4) and allow exogenous ATII cell engraftment (Figure 5 and 6).We also showed that without proper conditioning of the lung epithelium, even when defective, as in Sftpc - /-mice, cell engraftment is minimal, if not completely absent.While bleomycin fits the purpose of animal studies, we are aware that alternative conditioning methods need further investigation to be applicable to humans.
When considering cell therapy, the first question is the choice of engrafting cells.Recent studies have shown that murine primary, embryonic or pluripotent stem-cell derived cells can be transplanted in recipient murine lungs post injury and cells persisted up to 4-6 months 33,[56][57][58][59][60][61][62][63][64] .Human primary and pluripotent stem-cell derived cells have been transplanted into mice airways, and in one case, into distal lungs 58,60,62,65 .In all these previous studies, wild-type or immunodeficient mice were used as recipient; no previous models of cellular therapy have utilized an ATII cell disease models as in this study (see Supplemental Table 1).
Here, we used highly purified Sftpc +/+ ATII cells as a proof-of-concept study and a disease model as recipient.Transplanted primary ATII cells have previously been shown to promote a certain degree of lung recovery post-injury in wild-type mice 33,63 .The ATII cells transplanted in our study present several advantages.They are isolated from Sftpc +/+ mice lungs and they express SPC.Therefore, when transplanted in Sftpc -/-mice, those ATII cells can potentially correct the phenotype of the host, reintroducing SPC.They are easily identifiable in the Sftpc -/-by IF for pro-SPC and by RT-qPCR for Sftpc facilitating any engraftment analysis without requiring external manipulations for identification (e.g.lentiviral fluorescent labeling).Finally, since they are syngeneic, the host does not require any immunosuppression to allow engraftment.Multipotent Sox9 + murine lung progenitors have been isolated from fetal lungs and showed to engraft in immunocompromised mice after injury and differentiate in airway and alveolar cells 64 .Recently, another group has been able to derive and expand in vitro Nkx2-1 + /Sox9 + lung epithelial progenitors from murine embryonic stem cells (ESCs), called ESC-derived tip-like cells, and showed differentiation into ATII-like and ATI-like cells when transplanted into the lungs of syngeneic immunocompetent recipients 61 .While these results are promising, they have not yet been applied to a disease model nor evaluated for therapeutic effect on disease progression.
A second question is defining the number of defective cells to be replaced to demonstrate a therapeutic benefit for patients.Replacing all ATII cells may be neither safe nor needed.
For example, in Sftpb -/-mouse models, symptoms appear only when SPB levels fall below 20-25% of wild-type levels 66 .Our data support the idea that partial replacement of ATII cells is sufficient to promote lung repair, as we transplanted up to 1x10 6 ATII cells, one tenth of total ATII cell number in a mouse lung 67 , which was sufficient to attenuate lung injury secondary to bleomycin (Figure 6).
The third question is timing of intervention.All the current therapies of chILD aim to attenuate the downstream effects of the disease but do not target the main cell involved in its pathogenesis, the ATII cell 18,68 .In replacing or correcting defective ATII cells, timing is critical since it should ideally occur prior to irreversible chronic changes of the lung.In this study, we transplanted animals at 3 different ages (4, 8, and 12 mo) and used bleomycin as pre-conditioning strategy.We noticed that at 4 and 8 mo, low-dose of bleomycin allowed better engraftment among all the conditions tested (Supplementary Figure 7).In 12 mo mice, instead, cell engraftment was always lower for any bleomycin dose and variable between experiments respect to younger mice.This could be explained by the fact that the natural progression of lung disease of the Sftpc -/-mice aggravated by bleomycin creates a hostile milieu for cell attachment and engraftment.Our data suggested that cell therapy for progressive lung diseases such as chILD should occur early in the disease course.
We observed long-term engraftment and attenuation or repair of bleomycin-induced injury (Figure 6f-g).Our data support those of other groups on mouse-to-mouse cell transplants using bleomycin 33,57,58 .Louie and colleagues showed that Rag1 knock-out mice conditioned with bleomycin and transplanted with organoid cells derived from SCA1negative ATII cells had lower Ashcroft injury scores compared to those conditioned only with bleomycin 58 .Mice conditioned with bleomycin and transplanted with club-like lineage negative epithelial progenitors high in H2-K1 expression showed improved oxygen saturation at 2 weeks post-transplant 57 .In a similar study, mice transplanted with primary ATII cells post-bleomycin improved their oxygen saturation and their arterial partial pressure of oxygen at 2 weeks post-transplant 33 .Regardless of the mechanisms and signals involved, the current and other studies 33,57,58 showed that transplanted ATII or ATII-like cells could promote repair or functional recovery of the lung.We observed that one month after transplantation, mice receiving ATII cell transplant exhibited a marked improvement in LIS compared to both untreated mice (experiencing the natural progression of the disease) and mice treated with bleomycin (experiencing injury).
However, by the two-month mark, the disease improvement in ATII cell-transplanted mice was primarily noticeable in comparison to the mice injured with bleomycin (Figure 6).This might suggest that the disease naturally progresses faster than the regenerative capacity of transplanted cells.Future studies should explore whether transplanting subpopulations of ATII cells with high progenitor-capabilities (such as Wnt-responsive Axin2-expressing subset of ATII cell) 69,70 or murine ESC-derived tip-like cells 61 could yield prolonged and persistent benefits in chILD-like disease models.Nevertheless, a single cell transplant might not be adequate to entirely and permanently halt the progression of the disease and sequential cellular transplants might be necessary to effectively manage the disease over time.
Our study has several limitations.While bleomycin is commonly used as pre-conditioning strategy in animal models 33,57,58,61 , this conditioning might be challenging to apply in patients.Another limitation is that it is challenging to accurately determine the total number of engrafted Sftpc +/+ ATII cells in the entire mouse lung.While Sftpc +/+ ATII cells could be identified by pro-SPC immunostaining, the lack of lineage tracing of our transplanted cells did not allow us to follow the fate of these cells or determine if they differentiated into other lung cell types, including ATI cells.This could lead to underestimate the number of cells derived from the initially engrafted Sftpc +/+ ATII cells.
Nonetheless, our study demonstrates the successful engraftment, persistence of ATII cells, and their repairing capacity in a chILD-like disease model using low-dose bleomycin as conditioning strategy.Ultimately, Sftpc -/-mice with a minimal conditional strategy, such bleomycin, could represent a preclinical, translational platform to assess cell therapy in chILD.In conclusion, our study lays the foundation for cell therapy in chILD, offering an alternative approach to lung transplantation.

Methods
Animals.129S2/SvPasOrlRj (129/Sv) Sftpc -/-mice, generated by gene inactivation as previously described 39 , were generously donated by Dr. Whitsett, Cincinnati Children's Hospital.129/Sv Sftpc +/+ (wild-type) mice used as control group were purchased by Taconic Biosciences (NY, USA).In all the experiments, 4-, 8-, or 12-month-old mice, both female and male, were used.All animal work was approved by the Columbia University Institutional Animal Care and Use Committee and complied with the National Research Council Guide for the Care and Use of Laboratory Animals.Mice were humanely euthanized via inhalation of 5% isoflurane, followed by a second method of euthanasia: cervical dislocation followed by median sternotomy.Lungs were perfused via right ventricle with PBS (Phosphate Buffer Saline), and following perfusion, an incision was made, and the trachea was cannulated using a 20G catheter.The catheter was secured in position via a suture wire, and the lungs were filled by intratracheal instillation of 80% OCT (optimal cutting temperature compound, Sakura Finetek)/20% PBS, removed surgically from the animal, frozen, sectioned according to pre-determined map 42 and stored at -80°C.

Delivery of Bleomycin and Induction of Lung Injury. 15 Units (U) of bleomycin powder
(Meitheal Pharmaceuticals Inc.) was resuspended in 5 mL of sterile normal saline (NS) solution and stored sterile at 4°C.For any use, only the necessary amount was taken from the vial.4-12 months-old 129/Sv Sftpc +/+ and Sftpc -/-129/Sv mice were weighed and anesthetized with an intraperitoneal injection of Ketamine (80-95 mg/kg) and Xylazine (5-10 mg/kg).Intratracheal intubation was performed using a 20G cannula modified in length for murine lungs.Bleomycin was administered at 0.005, 0.01, or 0.05 U/mouse in a total volume of 40μL in sterile NS.Control mice received 40μL of vehicle (NS).Following i.t.administration, mice were allowed to recover from anesthesia and returned to their cages until euthanasia.Endpoint analysis was performed at 10 days from bleomycin injection.
Mice were humanely euthanized with isoflurane and lung tissues were harvested, frozen in OCT, and stored at -80°C.Tissues were processed for Hematoxylin and Eosin (H&E), immunofluorescent staining, or gDNA/mRNA extraction.
Stereological Analysis and Lung Injury Score.H&E sections were prepared by the Herbert Irving Cancer Center Molecular Pathology Core.Sections were scanned on a Leica AT2 slide scanner at 40X resolution (.25 microns/pixel).1000 x 1000 µm sections were randomly generated using a ImageJ macro as previously described 42 and then were analyzed for morphometric and stereological features, and LIS.For each analysis, 60-120 random sections were analyzed for each condition.Vvsep, Vsair, Lm, and Lmw were calculated according to the methods previously described 71,72 .Septal counts were performed manually by two independent researchers blind to the treatment group using the grid system previously described by our group 73 .For LIS, these same sections were analyzed by a pathologist blinded to the treatment group according to ATS guidelines 40,41 to determine lung injury 42 .
Hydroxyproline Assay.Hydryoxproline content was determined for each sample using Millipore Sigma Hydroxyproline Assay kit (MAK008).25-30 mg of tissue was sectioned from tissue-blocks cryopreserved in OCT.Sections were washed in PBS to remove residual OCT, weighed, dried, hydrolyzed, and ran according to manufacturer's protocol.
Isolation of ATII Cells.Healthy 129/Sv Sftpc +/+ mice at 2-3 months of age were humanely euthanized for isolation of Sftpc +/+ ATII cells.Lungs were perfused with PBS via the right ventricle of the heart and bronchioalveolar lavage was performed with PBS prior to inflate lungs in situ with Dispase (~0.9 ml, 50U/ml, Corning #354235) via tracheal cannulation.
Lungs were tied with suture wire and incubated in an additional 1 mL of Dispase at room temperature (RT) for 20 min.Following incubation, pulmonary lobes were separated from the trachea and bronchial tree and chopped mechanically with a variety of surgical scissors.After dissection and mechanical separation, the tissue was incubated at 37°C in MEM + DNase I with frequent agitation to complete digestion for 10 min.The mixed cell digest was filtered through 100 µm and 40 µm cup filters, and fibroblasts were removed from the suspension by three successive adherence steps on tissue-culture treated plastic.Alveolar epithelial type II cells were then purified from resident macrophages, lymphocytes, and blood cells through two passes of cell enrichment and isolation kits (Dynabeads DC Cell Enrichment Kit #114.29D;Dynal Mouse T Cell Negative Isolation Kit #114.13D).Kits were used as described in manufacturer protocol.The purity of the resulting ATII population was verified both via immunostaining (pro-SPC) after plating overnight the cells in Matrigel (1:30 mixed in media), and by selecting CD45-EpCAM+ population (CD45 BV421, Biolegend #103133; EpCAM PE/Cy7, Biolegend #118215) using a Sony MA900 cell sorter (San Jose, CA).
Bleomycin Conditioning and ATII Cell Engraftment.4-, 8-, or 12-months old Sftpc -/- 129/Sv mice were anesthetized and treated with 0.005-0.05U of bleomycin, or vehicle in 40µL volume via i.t delivery as previously described in this paper.Each age group contained 3 animals for each treatment condition.After 10 days, mice received 1x10 6 freshly isolated Sftpc +/+ ATII cells delivered i.t. and were harvested at 2-16 weeks post cell delivery.Lung tissues were inflated with 80% OCT/20% PBS and sectioned into upper, middle, and lower lobes according to a predetermined lung map that covers both lungs from upper to lower regions as previously described 42 .
Immunofluorescent Staining.Lung sections were thawed at RT for 5-10 minutes, fixed with 4% paraformaldehyde for 10 minutes at room temperature (RT) and washed with PBS for 5 minutes.The sections were permeabilized with 0.25% Triton X-100/PBS for 20 minutes followed by blocking in 10% donkey serum diluted in PBS for 1 hour.Primary antibodies (Supplementary Table 2) were diluted in 5% donkey serum in PBS and incubated at 4°C overnight.The following day, sections were washed with PBS + 0.025% Triton-X for three times, 5 minutes each followed by secondary antibody (Supplementary Table 3) incubation for 1 hour at RT.Following secondary incubation, sections were washed three times for 10 minutes with PBS + 0.025% Triton-X100 and finally mounted with DAPI contained fluorescent mounting medium.The following antibodies were used:  2 and 3).For proSPC and mature SPC staining in the transplant experiments analysis, TrueVIEW® Autofluorescence Quenching Kit (Vector, SP-8400-15) was applied and counterstained with DAPI prior to imaging.Samples were imaged using motorized DMi8 (Leica Microsystems, Buffalo Grove, IL) inverted microscopes.Confocal images were taken on a Leica Stellaris 8.For NKX2.1 stainings, 20 random 500 x 500 µm regions of interest were generated for each sample and positive cells were counted over total number of cells.
RT-qPCR.200 µm thick slices of lung tissue cryopreserved in OCT were sectioned and washed with PBS to remove residual OCT.For mRNA extraction, tissue was digest and homogenized in Trizol reagent according to manufacturer's protocol (Invitrogen™, #15596026).For RT-qPCR of mRNA expression, mRNA was extracted using Zymogen Direct-zol RNA Microprep kit (#R2062), and cDNA was generated from RNA extracts using the Multiscribe High-Capacity cDNA kit (ThermoFisher, #4368814).For the analysis, 20 ng of cDNA was analyzed per well.For genomic DNA extraction, tissue was lysed, and gDNA was extracted using Zymogen Quick-DNA Microprep Kit (#D3021) according to manufacturer's protocol.For the analysis, 100 ng gDNA was analyzed per well.DD Comparative qPCR was run on all samples using SYBR green reagents (ThermoFisher, #A25743) and using gene-specific PCR primers from Integrated DNA Technologies (Supplementary Table 4).

Data availability.
The data that support the findings of this study are available on request from the corresponding author, NVD.

Statistical methods.
Statistical methods applied for each experiment are outlined in the figure legends.Both male and female mice were used with no clear difference in response to bleomycin or transplantation.Unpaired, two-tailed Student's t tests were used when comparing two groups, while ANOVA and Tukey's test were used when comparing multiple groups.For each data set, mean±SEM were calculated and presented in the legend section of each figure.The differences between the groups were considered statistically significant for p £ 0.05.If not indicated, the difference between groups is not statistically significant.

Figure 2 .
Figure 2. Lung collagen content and ATII cell markers analysis in Sftpc -/-mice at

Figure 3 .
Figure 3. Susceptibility of Sftpc -/-mice to bleomycin.a. Schematics of the injury model